Continued analysis of the role of DNA synthesis, cell division and gene expression in the generation of cytotoxicity in vitro. a) MLC. We are using a new technique to form cell-cell conjugates between effector cells and target cells. By labeling one or the other with an intracellular fluorescent tag, we can get a very quantitative estimate of at least the relative proportion of effector cells on a day-to-day basis in a given population. We will use this approach, in conjunction with cell synchronization techniques, to ask whether the cytotoxicity per effector cell changes with subsequent rounds of cell division (after the first, which is a required one). b) Lectin-generated cytotoxicity; signals required for killer T cell triggering. We have shown that Con A is an effective inducer of antigen receptor-mediated cytotoxicity in both the primary and secondary MLC. While this is an important and interesting observation in itself, it has led us to look at the broader question of signals required for generating killer T cells. In the coming year, we will look at various modes of recalling the cytotoxic function in memory cells: lectins, cyclic nucleotides, AEF (supernates of MLCs or Con A activated cultures), third party cells, cyclic nucleotides, etc. Together with the studies on membrane organization in relation to the cytotoxic state (see below), these studies should give us considerable insight into the nature of effector T cell triggering requirements. T cell surface and membrane properties during generation of cytotoxicity. We are monitoring short-range changes in both fatty acid composition and fluidity after activation in both primary and secondary reactions using the various approaches to triggering described above. In addition, we are artificially manipulating the fatty acid composition of lymphocyte membranes in vitro, so that fluidity can be precisely controlled. We will examine the effects of altered membrane fluidity, as a function of temperature, on a number of parameters such as Con A binding, cytolysis, etc.